A collegue of mine wants to determine wether the parathormone produced by her cells is glycosylated or not. Does anybody know how to do this? Are there antibodies or ELISAs to test this?
She could try to see if her protein binds lectins. http://www.vectorlabs.com/catalog.aspx?catID=337
Thank you all for your answers so far.
We will need a method that can also quantify the amount of the different proteins.
MS is qualitative only, isn't it? And we want to analyse cell culture supernatants so i don't think Schiff is specific enough. The lectins may be worth a try. I'll have a look at the literature to see if there are any PTH-binding lectins.
Hi Juliane, MS will happily detect post translationally modified proteins either directly with applications such as electron transfer dissociation, or through conventional MS detection following enzymatic digestion of the protein (trypsin etc.) or the sugar (glucosidase) following some form of sample extraction. Effectively, any difference/change in mass or charge state is amenable.
It is correct that MS is dependent on the ionisation propensity of the analyte and therefore not inherently quantitative compared to techniques such as NMR, but if you are detecting a particular protein in a particular matrix, any one of several applications make quantification possible (labelling, internal standards etc.) http://en.wikipedia.org/wiki/Protein_mass_spectrometry
The solution is a combination between ELISA and a ligand assay.
Plate coating with an anti-PTH antibody. Plate washing, sample incubation as known from the normal ELISA. Now the differentiation. Use the second anti-PTH-HRP labeled antibody as before for one set of the samples. The second set of samples will be now incubated with a enzyme labeled lectin. The lectins has to be selected by the sugar residues at the hormone.
Reaction time and lectin concentrations need a small optimization. At the end you have both, the concentration of the whole enzyme and the proportion of the glycosylated. You need to find a standard hormone preparation fully glycosylated and one free of any glycosylation. An alternative could be the pretreatment with different glycosidases.
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